Nucleic acid modifying reactions play a pivotal role in modern biological and pharmaceutical research, both in the academic and industrial settings. Such reactions cover a wide range of applications, ranging from nucleic acid amplification reactions to regulated and specific cleavage of nucleic acids. These are mediated by enzymes that have been studied extensively for the past decades.
Amplification of target nucleic acid sequences is of importance to modern biological and pharmaceutical industry. Large-scale robotic facilities used in industrial research depend on the accurate and efficient regulation of amplification conditions to ensure that the target sequences are correctly amplified for downstream applications.
Regulation of the activity of such enzymes is however not a trivial task. In the case of polymerases, efficient amplification is dependent on a complex interplay of parameters such as primer length, GC content of both primer and target sequences as well as ionic strength and composition of the reaction buffer. Further, non-specific binding of primers is often observed at lower temperatures during the amplification cycles. This increases the fraction of non-specific side products and lowers the overall efficiency of the amplification reaction.
To address this, recent developments in the field of polymerases describe “Hot Start” polymerases. This class of enzymes is either chemically inactivated or has the active site blocked due to binding of a specific antibody or an aptamer. After an activation step at high temperature, the chemical modification is cleaved off and the enzyme is activated.
In addition to “Hot Start” polymerases, so called “Hot Start” primers and “Hot Start” nucleotides have also been developed. These are chemically modified primers, wherein the modification is cleaved off at high temperatures and thus the primer is rendered functional and is able to hybridize to its target sequence. However, synthesis of such primers is expensive and requires more time than standard primers. Both in the case of the “Hot Start” primer and polymerases, the blocking features are only available once since the heat-induced removal of the chemical modification is irreversible.
There is a need for methods that allow for the regulation of enzymatic activity without elaborate modification of enzymes and substrates.